Quantitative real-time PCR (qPCR) is a standard method used for quantification of specific gene expression. This utilizes either dsDNA binding dyes or probe based chemistry. While dsDNA binding dyes have the advantage of low cost and flexibility, fluorescence due to primer dimers also interferes with the fluorescence of the specific product. Sometimes it is difficult, if not impossible, to standardize conditions and redesign primers in such a way that only specific fluorescence of the products of test and reference genes are acquired. Normally, the fluorescence acquisition in qPCR using dsDNA binding dyes is done during the melting phase of the PCR at a temperature between the melting points of primer dimers and the specific product. We have modified the protocol to acquire fluorescence during the hybridization phase. This significantly increased the signal-to-noise ratio and enabled the use of dsDNA binding dyes for mRNA quantification in situations where it was not possible when measurement was done in the melting phase. We have demonstrated it for three mRNAs, E6, E7, and DNMT1 with β-actin as the reference gene, and for two miRNAs. This modification broadens the scope ofqPCR using dsDNA binding dyes.

Introduction

The techniques available to study mRNA expression are Northern blotting, quantitative real-time PCR, and microarrays. While Northern blotting is cumbersome, microarrays are used for high-throughput but less-sensitive analysis. Thus qPCR remains the gold standard for comparing gene expression levels (1,2,3). qPCR is done either using probe-based chemistry or dye-based chemistry. Though more specific, the main drawback of probes is the requirement of expensive gene-specific probes. Dye-based chemistry is comparatively economical as it requires only gene-specific primers and is generally the routine method. The dyes used include SYBR green, Syto 9, and EvaGreen which bind only to dsDNA. The main drawback of dye-based quantification is non-specific amplification or the formation of primer dimers, which also contribute to the increase in fluorescence and can thus decrease the signal-to-noise-ratio (SNR) where the fluorescence of the specific product is taken as the signal and the fluorescence of the primer dimer is taken as the noise.

Quantification of product is usually done by measuring fluorescence at a temperature above the melting temperature (T_m) of the nonspecific product, but below the T_m of the specific product (4). This can be defined as acquisition in the melting phase, since the temperature is increased above the extension temperature (usually 72°C) to a point where the primer dimers have melted but the specific product has not yet melted. However, primer dimer kinetics are seldom predictable, and on many occasions longer nonspecific products are formed with a T_m close to or almost overlapping that of the product, which interferes with the exact quantitation of the product alone. This cannot always be avoided by reducing the primer concentration, and sometimes constraints of template sequence preclude alternate primer design. This is especially true for quantitation of miRNAs. When gene expression studies are done using an internal reference gene, it is always better to choose a temperature for fluorescence measurement that avoids overlaps in the melting profile of the specific product and primer dimers of both target and reference genes, since it enables the amplification of both genes in one PCR run (5,6,7,8). Though not absolutely essential, it helps in avoiding run-to-run variations and repeated freeze-thaw cycles, and also saves on time. All these limit the use of dye-based chemistry in gene expression studies.

Instead of acquiring fluorescence in the melting phase, we have acquired fluorescence in the hybridization phase by including a step to denature the specific product and primer dimers and then decrease the temperature to a point where the specific product has hybridized but the primer dimers have not. We have investigated whether acquisition during the hybridization phase would improve the discrimination between the primer dimers and the specific products, and the SNR of the measurement. This was done for three mRNAs: human papilloma virus (HPV) genes E6 and E7, eukaryotic gene DNMT1, and small RNAs miR27 and U6 snRNA.

Materials and Methods

U87MG, SiHa, and MCF-7 cell lines (ATCC, Manassas, VA, USA) were used for experiments and were maintained in DMEM (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS (Sigma-Aldrich), 3.7 g/dL sodium bicarbonate, ciprofloxacin (10 µg/mL), and 5% CO₂ at 37°C. RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as per the manufacturer's protocol. miRNAs were isolated using the Purelink miRNA isolation kit (Invitrogen). After DNase (Ambion, Austin, Texas, USA) treatment, RNA was quantified using a spectrophotometer (Nano Drop ND-1000; Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis was done using 1 µg of RNA, RETROscript reverse transcriptase (RT) (Ambion), and random decamers. For miRNAs, cDNA synthesis was done using stem-loop primers (9).

Real-Time PCR

Quantitative PCR reactions and threshold value (Ct) calculations were done on a Rotor-Gene 6000 (Corbett Research, Mortlake, Australia) using the software provided. Amplification efficiencies of individual reactions were calculated using the Rotor-Gene 6000 (Corbett Research) software.

PCR reactions were carried in 10 µL reaction volumes: 2.5 µL of 1:5 diluted cDNA, 0.5 µL of primer mix (0.5 pmol/µL as final concentration of each primer), 1 µL of 10× Taq Buffer A, 0.5 U Taq DNA polymerase (both from Bangalore Genei, Bangalore, India), 1 µL Syto 9 (Invitrogen, Carlsbad, CA, USA), 0.25 µL of 10 mM dNTPs (Fermentas Inc, USA), and 4.6 µL of nuclease-free water (Ambion). In case of reactions involving standard curves, cDNA dilutions (x, x/2, x/4, and x/8) were prepared in nuclease-free water. All the samples were put up in triplicates. The real-time PCR products were run on an agarose gel to rule out any cDNA contamination in the no-template control (NTC) sample.